Hereditary Stability of an Engineered E. coli Strain DH5α/pCW-PL-XE-TNFαm2
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    Abstract:

    ObjectiveThe targeting fusion protein XE-TNFαm2 produced by the genetic engineered E. coli strain DH5α/pCW-PL-XE-TNFαm2 was shown a prospect to effectively eliminate the HIV in AIDS patients. Its expression level reached 32%~36% of total cell soluble proteins. The purpose of this study was to investigate the hereditary stability of this engineered strain. MethodsThis strain was transferred on LBAmp+ and LBAmp- solid culture media day by day as generation by generation for a total of 100 generations, and then stored at 4℃ for 4,5, 6 months, respectively. Finally, to check the protein XE-TNFαm2 (20.3 kDa) expression levels of each transferred generation strain on LBAmp+ and LBAmp- media cases, respectively, by general protocol for temperature control expression, so as to compare the expression levels in the above different cases. The samples were taken from generations transferred for 1,0, 0,0, 0,0, 0,0, 0,0, 100 days on LBamp+ and LBAmp-media, respectively. ResultsThe protein XE-TNFαm2 expression levels were not obviously changed from the 1st generation to 100th generation under LBamp+ and LBAmp- cases, respectively (P>0.05). Only in the case that after the strains were transferred for 100 generations on LBamp+ or LBAmp-media and then stored at 4℃ for 4,5 or 6 months, respectively, the protein XE-TNFαm2 expression levels were reduced by 8%. ConclusionsCIts857 sequence, PL promoter and termination T1T2 sequence, existing in this plasmid vector, are three key elements to maintain such a high expression level. This genetic engineering E. coli strain DH5α/pCW-PL-XE-TNFαm2 has good hereditary stability. 

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